SITE-SPECIFIC DEOXYNUCLEOTIDE SUBSTITUTIONS IN YEAST U6 SNRNA BLOCK SPLICING OF PRE-MESSENGER-RNA IN-VITRO

We have identified 2'-hydroxyl groups of the U6 phosphate-ribose backb one which are required for reconstitution of splicing activity in U6-d epleted yeast extract. To screen the 2'-hydroxyls of yeast U6 at nucle otides 39-88, spanning the conserved central domain, synthetic U6 RNAs were constructed with deoxyribonucleotides incorporated site specific ally. Only four individual deoxynucleotide substitutions blocked splic ing activity: dA51 (in the ACAGAG sequence), dA62 (next to the AGC tri ad), and dU70 and dC72 (both in the loop of the 3' intramolecular stem -loop), Native gel analysis revealed that these deoxy-substituted U6 R NAs were competent for assembly of spliceosomes, Interestingly, a 2'-O -methyl substituent at A51, A62, U70 or C72 did not inhibit splicing a ctivity, indicating that the essential 2'-OH groups at these positions in U6 act as hydrogen bond accepters or neutral coordinated ligands, The requisite 2'-hydroxyls at A62, U70 and C72 show both similarities and differences relative to the positions of essential 2'-hydroxyls of catalytic domain V of group II ribozymes. The identification of the e ssential 2'-hydroxyls at positions 62, 70 and 72 corroborates that the 3' intramolecular stem-loop in U6 plays an important role in pre-mRNA splicing.